MAMMALIAN THIOREDOXIN REDUCTASE IS IRREVERSIBLY INHIBITED BY DINITROHALOBENZENES BY ALKYLATION OF BOTH THE REDOX-ACTIVE SELENOCYSTEINE AND ITS NEIGHBORING CYSTEINE RESIDUE
J. Nordberg et al., MAMMALIAN THIOREDOXIN REDUCTASE IS IRREVERSIBLY INHIBITED BY DINITROHALOBENZENES BY ALKYLATION OF BOTH THE REDOX-ACTIVE SELENOCYSTEINE AND ITS NEIGHBORING CYSTEINE RESIDUE, The Journal of biological chemistry, 273(18), 1998, pp. 10835-10842
The immunostimulatory dinitrohalobenzene compound 1-chloro-2,4-dinitro
benzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase (
TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in
the modified enzyme (Arner, E. S. J., Bjornstedt, M., and Hohmgren, A.
(1995) J. Biol. Chem. 270, 3479-3482). Here we have further analyzed
the reactivity with the enzyme of DNCB and analogues with varying immu
nomodulatory properties. We have also identified the reactive residues
in bovine thioredoxin reductase, recently discovered to be a selenopr
otein. We found that 4-vinylpyridine competed with DNCB for inactivati
on of TrxR, with DNCB being about 10 times more efficient, and only al
kylation with DNCB but not with 4-vinylpyridine induced an NADPH oxida
se activity. A number of nonsensitizing DNCB analogues neither inactiv
ated the enzyme nor induced any NADPH oxidase activity. The NADPH oxid
ase activity of TrxR induced by dinitrohalobenzenes generated superoxi
de, as detected by reaction with epinephrine (the adrenochrome method)
. Addition of superoxide dismutase quenched this reaction and also sti
mulated the NADPH oxidase activity. By peptide analysis using mass spe
ctrometry and Edman degradation, both the cysteine and the selenocyste
ine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where
Sec indicates selenocysteine) were determined to be dinitrophenyl-alk
ylated upon incubation of native TrxR with NADPH and DNCB. A model for
the interaction between TrxR and dinitrohalobenzenes is proposed, inv
olving a functional FAD in the alkylated TrxR generating an anion nitr
oradical in a dinitrophenyl group, which in turn reacts with oxygen to
generate superoxide. Production of reactive oxygen species and inhibi
ted reduction of thioredoxin by the modified thioredoxin reductase aft
er reaction with dinitrohalobenzenes may play a major role in the infl
ammatory reactions provoked by these compounds.